The cyclic nucleotide phosphodiesterase and its inhibitor are secreted glycoproteins that control extracellular levels of cAMP during the development of Dictyostelium discoideum. Both of these molecules were purified in this laboratory. We have now cloned their genes and analysed the structure and the regulation of each. The cyclic nucleotide phosphodiesterase (PDE) gene is the most complex yet characterized in this organism and mediates chemotactic and gene regulation events at all stages of the developmental cycle. The inhibitor gene is expressed only during the aggregation phase of development. We will continue our studies of the regulatory system constituted by the PDE and the inhibitor. A question that is unsolved is the mechanism by which the PDE is attached to the external face of the plasma membrane during the aggregation phase of development. We will test a model that predicts an unusual form of attachment. Once the mechanism is understood we will study the biological consequences of modifying the attachment. The discovery of a third promoter that is active only during late development and only in prestalk cells has led to the analysis of the role of cAMP and adenosine in cell type proportioning during post-aggregation development. Each promoter will be fused to beta-galactosidase to determine its site of activity at all stages of development. We will then use the PDE, the inhibitor and other genes fused to various promoters to transform cells and regulate the concentrations of extracellular cAMP and adenosine during post-aggregation morphogenesis. The aggregation specific and the stalk cell specific promoters of the PDE gene and the promoter of the inhibitor gene will be analysed and the biological effects of altering them will be studied.